Method of preparation of purified agar



TABLE 1 Percent Glutamic Biotin Trans Acid, g./l.

rmssion Deficient 75 10. 1 37 26. 6 29 32. 2 Excessive h 21 8. 7

Example II The nitric acid-hydrolyzed starch solution of Example I is utilized as the carbohydrate source in the nutrient medium used in a fermentation for the production of glutamic acid following essentially the same procedure used in Example I except a glutamic acid-producing strain of the organism Micrococcus glutamicus is employed.

Example III The nitric acid-hydrolyzed starch solution of Example I is utilized as the carbohydrate source in the nutrient medium used in a fermentation for the production of glutamic acid following essentially the same procedure used in Example I except a glutamic acid-producing strain or the organism Bacillus subtllus is used.

Example IV The nitric acid-hydrolyzed starch solution of Example I is utilized as the carbohydrate source in the nutrient medium utilized in a fermentation for the production of glutamic acid following essentially the same procedure used in Example I except an acid-producing strain of the organism Micrococcus varians is used.

Example V to Example VII The nitric acid-hydrolyzed starch solution of Example I is utilized as the carbohydrate source in the nutrient medium utilized in a fermentation for the production of glutamic acid following essentially the same procedure used in Example I except a glutamic acid-producing strain of the organisms Brevibacterium divarica-zum NRRL- 2312, Microbacterium flavum, Corynebacterizrm lilium, and Arthrobacter glofiformis, respectively, are used instead of Brevibacterium divaricatum NRRL-2311.

It is claimed:

1. A process for the production of glutamic acid by fermentation which comprises hydrolyzing in the presence of nitric acid a starch source containing more than 5 mcg. of biotin per 100 g. of carbohydrate, incorporating said hydrolyzed starch in a nutrient fermentation medium,

cultivating a glutamic acid-producing strain of the organism Brevibacterium divaricatum in said fermentation medium and recovering glutamic acid from said medium.

2. A process for the production of glutamic acid by fermentation which comprises hydrolyzing a starch source containing more than 5 mcg. of biotin per g. of carbohydrate in the presence of nitric acid for a length of time sufficient to hydrolyze the starch in said starch source to sugar whereby substantially all of the biotin is inactivated, neutralizing said hydrolyzed starch to pH 7.0, incorporating said hydrolyzed starch in a nutrient fermentation medium comprising a nitrogen source, a potassium source, a phosphate source, a biotin source and trace amounts of mineral salts, cultivating a glutamic acidproducing strain of the organism Brevibacterium divaricatum in said fermentation medium and recovering glutamic acid from said medium.

3. The process of claim 2 wherein the Brevibacterizmz divarlaczam is NRRL B2312.

4. The process of claim 2 wherein the Brevz'bacterium divaricatum is NRRL 13-2311.

5. A process for the production of glutamic acid by fermentation which comprises hydrolyzing a starch source containing more than 5 mcg. of biotin per 100 g. of carbohydrate in the presence of nitric acid, incorporating said hydrolyzed starch source in a nutrient fermentation medium, cultivating a glutamic acid-producing microorganism in said nutrient fermentation medium and recovering glutamic acid from said medium.

6. A process for the production of glutamic acid by fermentation which comprises hydrolyzing starch containing more than 5 mcg. of biotin per 100 g. of carbohydrate in the presence of nitric acid in an amount and for a time sufficient to hydrolyze said starch whereby the biotin content is lowered to less than 5 mcg. per 100 g. of carbohydrate, incorporating said hydrolyzed starch in a nutrient fermentation medium, and cultivating a glutamic acid-producing microorganism in said hydrolyzed starch containing nutrient fermentation medium to produce glutamic acid and recovering glutamic acid from said medium.

7. The process of claim 6 wherein said microorganism is selected from the group consisting of the glutamic acidproducing strains of the genera Bacillus, Micrococcus, Microbacterium, Brevibacterium, Corynebacterium, and Arthrobacter.

8. In a process for the production of glutamic acid by fermentation comprising cultivating a glutamic acid-producing microorganism in a nutrient fermentation medium containing a carbohydrate source to produce glutamic acid, and recovering glutamic acid from said medium, that step comprising incorporating in said fermentation medium as the said carbohydrate source hydrolyzed starch having before hydrolysis a biotin content of more than 5 mcg. of biotin per 100 g. of carbohydrate, said hydrolyzed starch having been hydrolyzed in the presence of nitric acid whereby the biotin content was reduced to less than 5 mcg. per 100 g. of carbohydrate.

No references cited.

LIONEL M. SHAPIRO, Primary Examiner.

United States Patent 3,362,885 PROCESS FOR THE PRODUCTION OF GLUTAMIC ACID Roger L. Harned, Terre Haute, Ind., assignor to Commercial Solvents Corporation, New York, N.Y., a corporation of Maryland No Drawing. Filed July 19, 1965, Ser. No. 473,232

8 Claims. (Cl. 195-47) This invention generally relates to a process for the production of glutamic acid by fermentation. In a particular aspect it relates to an improved nutrient medium for production of glutamic acid by fermentation.

Glutamic acid is produced in high yields by the fermentation of nutrient media with certain glutamic acid-producing strains of microorganisms. A satisfactory method for the production of glutamic acid is described in US. Patent No. 2,978,383 and US. Patent No. 2,978,384, both issued on Apr. 4, 1961, to Koichi Yamada. This method utilizes an aqueous nutrient medium comprising a carbohydrate source, a nitrogen source, a phosphate source, a potassium source, and trace amounts of mineral salts. Briefly, the process for the production of glutamic acid involves incubating a glutamic acid-producing strain of the microorganisms, Brevibacterium divaricazum', in an aqueous nutrient medium containing the above-mentioned ingredients. The fermentation is preferably carried out at temperatures ranging from about 30 to about 40 C. at a pH ranging from about 6 to about 9 under submerged conditions of agitation and aeration. The organisms of the genera Bacillus, Micrococcus, Brevibacterium, Microbacterium, Corynebacterium, Arthrobacter, and the like may also be used to produce glutamic acid by similar processes using similar nutrient media as are described in the art.

Acid-hydrolyzed starch is an excellent carbohyrate source for nutrient media used in fermentation processes, and especially in nutrient media used for the production of glutamic acid by fermentation. In the processes previously used, crude or refined starch is hydrolyzed in the presence of sulfuric acid to convert the starch to reducing sugar, usually calculated as glucose. Refined starch is relatively expensive compared with crude starch sources, such as ground corn flour and dry-milled starch obtained by dry milling of cereal grains, but these crude starch sources are not satisfactory for glutamic acid production by fermentation because they contain an undesirable amount of biotin, which is not aifected by the acid treatment heretofore used. In general, an amount of biotin of more than meg/100 g. of the carbohyrate content of the crude starch is undesirable for glutamic acid production because it leads to prolific growth of the organism without concomitant production of glutamic acid. It is known, however, that biotin in an amount of from about 1 mcg. to 5 mcg. per 100 g. of carbohydrate is beneficial for the production of glutamic acid, as disclosed by Kinoshita in US. Patent 3,002,889. The sulfuric acid hydrolyzate obtained from crude starch contains biotin in such amounts that when the hydrolyzate is employed as the carbohydrate source in the nutrient medium for the production of glutamic acid by fermentation, little or no glutamic acid is produced.

Previous workers have also been concerned with the problem of excess biotin in carbohydrate sources. Okumura et al., US. Patent 3,136,702, solved the problem by treating the carbohydrate source with a yeast and then employing the treated carbohydrate source in a nutrient medium. Okada et al., US. Patent 3,138,540, treated cane and beet sugar molasses with hydrogen peroxide or ammonium persulfate to simultaneously decolorize the molasses and remove the excess biotin. These procedures were intended for saccharide materials and there Patented Jan. 9, 1968 ice is no indication that they would be satifactory for treating acid-hydrolyzed crude starch.

It is an object of this invention to provide a new process for the production of glutamic acid.

It is an additional object of this invention to provide a new carbohydrate source for nutrient media used in glutamic acid fermentation processes.

It is another object of this invention to provide an improved nutrient medium for the production of glutamic acid by fermentation.

Still another object is to provide a carbohydrate source substantially free from biotin for use in a nutrient medium for production of glutamic acid by fermentation.

It is a further object of this invention to provide a method for using a crude starch material containing an undesirably high amount of biotin in a glutamic acid fermentation process.

Other objects will be apparent to those skilled in the art from the description of this invention.

A new process has been discovered for the production of glutamic acid by fermentation which comprises hydrolyzing a starch source containing more than 5 meg. biotin per 100 g. carbohydrate in the presence of nitric acid, incorporating the hydrolyzed starch source in a fermentation medium, cultivating a glutamic acid-producing microorganism in said fermentation medium and recovering glutamic acid from said medium. By means of the new process the above-mentioned problems of the hydrolyzed biotin-containing starch are lessened. Therefore, in the new process, it is possible to use the higherbiotin starch sources as raw materials. Because the higher biotin starch sources are generally more economical than refined starch sources, and because hydrolysis in the presence of nitric acid eliminates unsatisfactory fermentation due to excess biotin, the nutrient medium provided by the present process represents an improvement over previous nutrient media used for the production of glutamic acid by fermentation using glutamic acid-producing strains of microorganisms, e.g., Micrococcus glutamz'cus, Brevibaczerium divaricatum, and the like.

Generally, the process consists of hydrolyzing a crude starch source containing more than 5 mcg. of biotin per 100 g. carbohydrate in the presence of nitric acid in an amount and concentration and for a time period efiective to convert substantially all of the starch to sugar whereby the biotin level is reduced to below 5 meg/100 g. of carbohydrate. A preferred process consists of partially hydrolyzing with stirring at first increment of a viscous, aqueous suspension of a starch source contaming more than 5 mcg. per 100 g. of carbohydrate in the presence of nitric acid at a temperature of about C. until the suspension thins, adding with stirring a second increment of said starch suspension, again heating at 80 C. until the suspension thins, and then completing the hydrolysis by heating under steam pressure for 2 hours in an autoclave at 15 p.s.i.g. After heating, the hydrolyzate is cooled, neutralized with ammonium hydroxide and filtered. The resultant hydrolyzed starch will now contain less than 5 mcg. biotin per g. carbohydrate. It is then used as the carbohydrate source in a nutrient medium for the production of glutamic acid by fermentation in accordance with methods known in the art, e.g., the method described in US. Patents 2,978; 383 and 2,978,384.

The heating period for hydrolysis is not critical, but a longer period is required at low temperatures than at high. The hydrolysis of starches with sulfuric acid is a procedure well known in the art and, in general, the same conditions are used for the hydrolysis of starch with nitric acid in accordance with this invention. A period of about 15-30 minutes at a temperature of about 80 C. is generally adequate for the preliminary treat- 

1. THE METHOD FOR PREPARATION OF A PURIFIED COLDWATER INSOLUBLE, NEUTRAL GALACTOSE POLYMERIC CONSTITUENT AND, IN ADDITION, CONTAINS CALCIUM AND A COLDWATER INSOLUBLE, SULFATED POLYSACCHARIDE COLD-WATER SOLUBLE BY ADDING TO THE AGAR, UNDER AQUEOUS CONDITIONS, A SOLUBILIZING AGENT SELECTED FROM THE GROUP CONSISTING OF PECTINASE ENZYME AND CALCIUM SEQUESTERANT, AND THEREAFTER SEPARATING THE COLD-WATER INSOLUBLE CONSTITUENT FROM THE COLD-WATER SOLUBILIZED SULFATED POLYSACCHARIDE. 